Seasonality, Composition, and Antioxidant Capacity of Limonene/δ-3-Carene/(E)-Caryophyllene Schinus terebinthifolia Essential Oil Chemotype from the Brazilian Amazon: A Chemometric Approach

Schinus terebinthifolia Raddi is widely used in traditional Brazilian medicine to treat respiratory diseases, as an antiseptic, anti-inflammatory, and hemostatic agent. This study aimed to evaluate the influence of climatic parameters on the yield, antioxidative capacity, and chemical composition of the S. terebinthifolia leaf essential oil. The specimen was collected monthly from October 2021 to September 2022. Leaf essential oils (EOs) were obtained by hydrodistillation, and their chemical compositions were analyzed by gas chromatography/mass spectrometry (GC/MS). Statistical analyses were performed to verify the climatic influences on the yields, chemical composition, and antioxidative capacity. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging and inhibition of β-carotene/linoleic acid oxidation assays were performed to assess the antioxidant activity. The leaf essential oil yields ranged from 0.1% (July) to 0.7% (May and September), averaging 0.5 ± 0.2%. There was no significant difference in essential oil production during the dry (0.4 ± 0.2%) and rainy (0.6 ± 0.1%) seasons. The main chemical constituents identified in essential oils were limonene (11.42–56.24%), δ-3-carene (8.70–33.16%) and (E)-caryophyllene (4.10–24.98%). The limonene annual average was 43.57 ± 12.74% and showed no statistical difference during the dry (40.53 ± 13.38%) and rainy (52.68 ± 3.27%) seasons. Likewise, the annual average of δ-3-carene was 22.55 ± 7.11%, displaying no statistical difference between dry (26.35 ± 7.90%) and rainy (31.14 ± 1.63%) seasons. The annual average of (E)-caryophyllene was 11.07 ± 7.15% and this constituent did not show a statistical difference in Tukey’s test (p > 0.05) during the dry (12.72 ± 7.56%) and rainy (6.10 ± 1.78%) season. Limonene showed a moderate positive and significant correlation (p < 0.05) with precipitation (r = 0.56) and a weak correlation with temperature (r = −0.40), humidity (r = 0.40), and insolation (r = −0.44). All samples inhibited the oxidation in the β-carotene/linoleic acid system (22.78–44.15%) but displayed no activity in the DPPH method.


Introduction
The Anacardiaceae includes 79 genera with economic potential for providing resins, tannins, and edible fruits such as cashew (Anacardium occidentale L.) and mango (Mangifera indica L.) [1]. The Anacardiaceae genera are subdivided into five tribes: Anacardieae, Dobineae, Rhoeae, Semecarpeae, and Spondiadeae. Approximately 25% of this family's genera are known to be toxic, and these are limited to the tribes Anacardieae, Rhoeae, and Semecarpeae. Moreover, phytochemical and biological studies have only been performed on less than 7% of the known Anacardiaceae species [2]. Many taxa are also cultivated as ornamentals, such as the Schinus genus [3].
The Schinus genus has approximately 37 species, most native to South America [4]. Schinus plants are dioecious and female trees are rich sources of potentially active compounds of several secondary metabolites, such as flavonoids, biflavonoids, tannins, catechins, triterpenes, steroids, and essential oils [5]. Many plants in this genus are used in traditional medicine for various diseases, including rheumatism, bronchitis, hypertension, ulcers, abdominal pain, menstrual disorders, gonorrhea, bronchitis, conjunctivitis, dysentery, urinary tract disorders, and eye infections [5].
Schinus terebinthifolia Raddi is known as "aroeira-vermelha", "aroeira-pimenteira", "Brazilian pepper", or "pink-pepper" [6]. This tree is native to Paraguay, northeastern Argentina, and Brazil and has been introduced in subtropical areas worldwide [7]. S. terebinthifolia leaves contain lanceolate and pointed leaflets, its small flowers are arranged in white or greenish-yellow pedicles, and its fruit is a red drupe with an aroma similar to that of pepper [8]. Moreover, the dried fruit is sold commercially as "pink pepper" [9] and the fruit essential oil (Schinus molle L.) is also commercially available [10]. This species is widely used in traditional Brazilian medicine. The leaf is used as an antiseptic, antiinflammatory, and hemostatic agent [11], and a leaf infusion is used to treat respiratory diseases [12]. In addition, some in-vitro and in-vivo studies have reported biological activities of S. terebinthifolia leaf and root extracts such as cytotoxicity against cancer cell lines [13], antioxidant [14], bactericidal, and fungicidal [15,16].
The chemical compositions of essential oils of Schinus terebinthifolia have already been described in the literature, presenting germacrene D (33.80%) and (E)-caryophyllene (12.25%) as main constituents [12]. However the chemical composition of essential oils is variable, depending on the analyzed plant part, origin, season, and extraction methods, as secondary metabolites can have their biosynthesis affected by natural processes such as plant development, rainfall, seasonality, and temperature of the environment, among other factors that influence the concentration of active constituents [17].
Therefore, considering the chemical and biological potential of S. terebinthifolia, this work is aimed to evaluate the influence of seasonality on yield, chemical composition, and antioxidant capacity of a limonene/δ-3-carene/(E)-caryophyllene Schinus terebinthifolia leaf essential oil chemotype from the Brazilian Amazon. In this seasonal investigation, the S. terebinthifolia leaf essential oil yields (v/w) ranged from 0.1% (July) to 0.7% (May and September), with an average of 0.5 ± 0.2% during the year of investigation. Statistically (Tukey test), no significant differences in essential oil yield were observed comparing the dry (0.4 ± 0.2%) and rainy (0.6 ± 0.1%) seasons. With respect to the relationship between essential oil yield and climatic parameters, either insignificant or minor correlations were discerned (p > 0.05) between the essential oil yield and humidity (r = 0.19), temperature (r = -0.22), and insolation (r = -0.26); precipitation (r = 0.43) displayed weak correlation with essential oil yield (Table 1). The leaf and fruit essential oil yields of S. terebinthifolia sampled in Rio Grande do Sul (southern Brazil) were 0.74 and 0.16% v/w, respectively [3]. Moreover, the essential oil yield of S. terebinthifolius leaves sampled in Minas Gerais State (southeastern Brazil) showed minor changes throughout one year. The essential oil yield ranged from 0.65 to 0.69% in the months of March to September, and 0.45 to 0.55% from October to February, which concurred with the flowering and fruiting stages, respectively [12]. In this study, the average yield was 0.46 ± 0.11% during the flowering stage (October to February, and September) and 0.45 ± 0.2% during the fruiting stage (March to August), showing no significant difference. However, in another specimen collected in El Ghazala, northern In this seasonal investigation, the S. terebinthifolia leaf essential oil yields (v/w) ranged from 0.1% (July) to 0.7% (May and September), with an average of 0.5 ± 0.2% during the year of investigation. Statistically (Tukey test), no significant differences in essential oil yield were observed comparing the dry (0.4 ± 0.2%) and rainy (0.6 ± 0.1%) seasons. With respect to the relationship between essential oil yield and climatic parameters, either insignificant or minor correlations were discerned (p > 0.05) between the essential oil yield and humidity (r = 0.19), temperature (r = −0.22), and insolation (r = −0.26); precipitation (r = 0.43) displayed weak correlation with essential oil yield (Table 1). The leaf and fruit essential oil yields of S. terebinthifolia sampled in Rio Grande do Sul (southern Brazil) were 0.74 and 0.16% v/w, respectively [3]. Moreover, the essential oil yield of S. terebinthifolius leaves sampled in Minas Gerais State (southeastern Brazil) showed minor changes throughout one year. The essential oil yield ranged from 0.65 to 0.69% in the months of March to September, and 0.45 to 0.55% from October to February, which concurred with the flowering and fruiting stages, respectively [12]. In this study, the average yield was 0.46 ± 0.11% during the flowering stage (October to February, and September) and 0.45 ± 0.2% during the fruiting stage (March to August), showing no significant difference. However, in another specimen collected in El Ghazala, northern Tunisia, the leaves presented a yield of 1.06% (w/w on dry weight) [20]. On the other hand, Santana et al. [21] collected the fresh leaves of S. terebinthifolia in Diadema City, São Paulo (southeastern Brazil), and obtained 571 mg of crude essential oil (yield of 0.17%).

Seasonal Effects in Essential Oil Chemical Composition
The 52 volatile components in the essential oils of the leaves of S. terebinthifolia, identified by GC/MS and quantified by GC-FID, are presented in Table 2, which are listed in order of their elution from the GC. The identified components comprise 98.06-99.93% of the total essential oil compositions in this seasonal investigation.

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Limonene and (E)-caryophyllene arise from different biosynthetic cations ( Figure 5) which explains why in July, for example, (E)-caryophyllene presented the major conten while limonene presented the lower content, the opposite happened in May. The same thing happened to δ-3-carene and (E)-caryophyllene; in July, there was a lowe concentration of δ-3-carene and a higher concentration of (E)-caryophyllene; February presented the higher content of δ-3-carene and a low content of (E)-caryophyllene. On the same line of thought, limonene and δ-3-carene arise from to same biosynthetic precurso cation, it is noticeable that from October to November the content of δ-3-carene increases and limonene decreases. Moreover, α-copaene and β-selinene belong to the biosynthetic precursor of germacryl cation.

Antioxidant Capacity
The antioxidant capacities of the essential oil samples were evaluated using two different assays. All samples inhibited the oxidation in the β-carotene/linoleic acid system (22.78-44.15%) ( Table 3), while the DPPH radical scavenging assay showed no inhibition The greater inhibition was observed in the essential oil sampled in August (44.15 ± 3.0%) and June (37.62 ± 2.74%), with only half of the Trolox standard inhibition (82.93 ± Figure 5. Biosynthetic pathway of (E)-caryophyllene, δ-3-carene and limonene.

Antioxidant Capacity
The antioxidant capacities of the essential oil samples were evaluated using two different assays. All samples inhibited the oxidation in the β-carotene/linoleic acid system (22.78-44.15%) ( Table 3), while the DPPH radical scavenging assay showed no inhibition. The greater inhibition was observed in the essential oil sampled in August (44.15 ± 3.0%) and June (37.62 ± 2.74%), with only half of the Trolox standard inhibition (82.93 ± 1.8%). The months of October to May showed no statistical difference in the Tukey test (p > 0.05), with inhibition between 22.77 to 30.65%. Hassimotto et al. defined that a percentage of oxidation inhibition between 40 and 70% characterizes an intermediate antioxidant capacity [28]. Further-more, there was a weak correlation between limonene amounts and a negligible correlation between δ-3-carene and (E)-caryophyllene and antioxidant capacity.
In the β-carotene/linoleic acid assay, β-carotene rapidly changes color in the absence of antioxidants. This is due to the coupled oxidation of β-carotene and linoleic acid, which generates free radicals. Formed by abstracting a hydrogen atom from its diallylmethylene group, the linoleic acid radical attacks a highly unsaturated β-carotene molecule. As a result, β-carotene is oxidized and partially degraded, subsequently losing its chromophore and its characteristic orange color [29]. However, the DPPH assay is based on radical scavenging; when a compound that can donate a hydrogen atom is mixed with a solution of DPPH, the DPPH radical is reduced with concomitant loss of the violet color, then, the free radical formed can undergo additional reactions to create a stable product. While DPPH can either accept a hydrogen atom or an electron to form a stable, diamagnetic molecule, and oxidation of DPPH is difficult and irreversible [30].
Monoterpene-rich extracts have demonstrated antioxidant activity against DPPH, although, when only limonene was tested, it was less reactive [31]. Different concentrations of δ-3-carene were tested in the DPPH assay, the higher inhibition (4.8 ± 0.4%) occurred at 4 µg/mL, showing low activity [32]. (E)-Caryophyllene showed a weak antioxidant efficacy in the DPPH method (IC 50 132.0 ± 9.9 µg/mL); however it was effective in antioxidant chain breaking in lipid peroxidation in vitro and had greater radical-scavenging behavior with reactive oxygen species than with relatively stable organic radicals [33]. Therefore, the low antioxidant capacity of Schinus terebinthifolia essential oil can be rationalized by the low capacity of the major components.

Plant Material and Climatic Data
The leaves of Schinus terebinthifolia were collected from a single specimen in Belém city, Pará state, Brazil (coordinates: 1 • 27 13.4 S/48 • 29 34.1 W). For the seasonal study, the mature leaves (150 g) were sampled on day 30 of each month, at 3 pm, from October 2021 to September 2022. Plant identification was performed by comparison with an authentic specimen of Schinus terebinthifolia Raddi, and a plant sample was deposited with the Herbarium "João Murça Pires", at Museu Paraense Emílio Goeldi, Belém city, State of Pará, Brazil (MG-245400). The specimen was collected in agreement with Brazilian laws concerning biodiversity protection (A075D38).
During the collection period, the climatic parameters (insolation, relative air humidity, and rainfall precipitation) of the collection site were obtained each month from the website of the Instituto Nacional de (

Extraction and Essential Oil Composition
The leaves of S. terebinthifolia were air-dried and 150-g samples were pulverized and hydrodistillation using a Clevenger-type apparatus for 3 h. The hydrodistillation was repeated twice for each sample. The essential oils were dried over anhydrous sodium sulfate, and the masses of dry plant material were used to calculate the essential oil yields. The moisture content of the plant samples was determined using an infrared moisture balance for water loss measurement. Analysis of essential oil yield was conducted in duplicate. The essential oil was dissolved in n-hexane (1500 µg/mL, 3:500, v/v) and analyzed by gas chromatography-flame ionization detector (GC-FID, Shimadzu Corporation, Tokyo, Japan) and gas chromatography-mass spectrometry (GC/MS, Shimadzu Corporation, Tokyo, Japan) simultaneously using the two systems. The essential oil analyses were performed in a GCMS-QP2010 system (Shimadzu Corporation, Tokyo, Japan), equipped with an AOC-20i auto-injector and the GCMSSolution software that included both the Adams and FFNSC-2 libraries [26,27]. The GC column used was an Rxi-5ms (30 m; 0.25 mm; 0.25 µm film thickness) silica capillary column (Restek Corporation, Bellefonte, PA, USA). The following operating conditions for the analysis were injector temperature = 250 • C; oven temperature programming was 60-250 • C at a rate of 3 • C/min); helium was used as the carrier gas, which was set to a linear velocity of 36.5 cm/s (1.0 mL/min); 1.0 µL of essential oil solution (6 µg of essential oil injected) was injected using a splitless mode of injection; ionization by electronic impact at 70 eV; the ionization source temperature was 220 • C and the transfer line temperature was 250 • C. The mass spectra were obtained using a scan range of 40-450 m/z and a scan rate of 2.0 scans/sec. The retention indices were calculated for all volatile components based on a homologous series of C8-C40 n-alkanes (Sigma-Aldrich, Milwaukee, WI, USA), according to the linear equation of van Den Dool and Kratz [35]. Each Individual component was identified by comparing its retention index and mass spectral and fragmentation pattern with those found in the GCMS-Solution system libraries. The quantitative data regarding the volatile constituents were obtained using a GC 2010 Series instrument with a flame ionization detector, operated under similar conditions to the GC-MS system, detector temperature of 250 • C. The percent compositions of individual components were calculated by peak-area normalization without a response factor using the flame ionization detector (GC-FID). The GC-FID and GC/MS analyses were carried out in duplicate.

β-Carotene/Linoleic Acid Assay
The stock solution of β-carotene/linoleic acid mixture was prepared by dissolving 0.2 mg of β-carotene in 1 mL of HPLC grade chloroform, followed by the addition of 20 µL of linoleic acid and 200 mg of Tween 20. The chloroform was then completely evaporated under reduced pressure. Then, 50 mL of oxygenated water was added with vigorous agitation. Aliquots (2500 µL) of the β-carotene/linoleic acid reaction mixture were distributed into test tubes and 200-µL portions of the essential oil samples (1.0 mg/mL in ethanol) were added. The emulsion systems were incubated at 50 • C. The same procedure was carried out using Trolox and a blank of ethanol as the control. The absorbances of the solutions were recorded at 470 nm and monitored at intervals of 15 min, for 120 min. The antioxidant activity (AA%) was calculated as the percent inhibition relative to the control using AA% = 1 − Abs 0 sample − Abs 120 sample / Abs 0 control − Abs 120 control × 100. All tests were carried out in triplicate [36].

Statistical Analysis
Statistical significance was evaluated using the Tukey test (p < 0.05). Pearson correlation analyses were carried out to determine the relationship between the major es-sential oil components (δ-3-carene, limonene, α-copaene, (E)-caryophyllene, α-humulene, γ-muurolene, β-selinene, α-selinene, and δ-cadinene) and the climatic parameters analyzed (insolation, relative air humidity, temperature, and rainfall precipitation), using the software GraphPad Prism, version 5.0. The principal component analysis (PCA) was utilized to verify the interrelation in the essential oil components (>2.0%) using the Minitab ® software (free 390 Version, Minitab Inc., State College, PA, USA). The hierarchical cluster analysis (HCA) was carried out using the Euclidean distance and Ward linkage to verify the similarity of the essential oil samples based on the distribution of the constituents selected in the previous PCA analysis [38].

Conclusions
The Schinus terebinthifolia essential oil yield is not correlated with climatic parameters, showing no statistical difference between the rainy and dry seasons. Limonene and δ-3carene were the main compounds throughout the study period, except in July, when the main constituent was (E)-caryophyllene, with quantitative variations in their concentration, which characterize a chemotype yet not described in the literature.
Moreover, all the samples inhibited the oxidation in the β-carotene/linoleic acid system and there was a weak or negligible correlation between limonene and δ-3-carene amounts and antioxidant capacity.
Thus, the variation in the content of the main constituents was not explained/correlated to the climatic parameters. Since there were quantitative and qualitative variations in the chemical composition of S. terebinthifolia essential oil, future studies focusing on seasonality, comparison between different plant tissues, antifungal, antibacterial, and other biological activities would be informative. A prior understanding of the phytochemical variations of the plant is necessary to appreciate the medicinal utility of S. terebinthifolia.  Acknowledgments: The student, B.d.A.G. is grateful to FAPESPA for a scientific initiation scholarship.

Conflicts of Interest:
The authors declare no conflict of interest.

GC/MS
Gas chromatography-mass spectrometry GC-FID Gas chromatography-flame ionization detector HCA Hierarchical cluster analysis PCA Principal component analysis R Pearson's correlation coefficient RI (C) Calculated Retention Index RI (L) Literature Retention Index